Insulin-stimulated redistribution of the GLUT4 glucose transporter from intracellular compartments to the surface of fat and muscle cells is the major mechanism for regulating postprandial glucose disposal and storage. This translocation is defective in Type II diabetes, although the molecular defect(s) that cause insensitivity to insulin is not known. It is important to understand both the mechanisms responsible for the basal state intracellular retention of GLUT4 and for the insulin-induced translocation to the surface. In this renewal application we continue our studies of insulin-regulated trafficking in 3T3-L1 adipocytes. The long-term objective is to understand this specialized trafficking pathway at a molecular level. In this project we will exploit the technical advantages of 2 reporter molecules that we have developed: a GLUT4-GFP construct and a chimera between IRAP and the transferrin receptor (vpTR). In Aim 1 of this application we will test a two-step insulin-regulated retention model of GLUT4 trafficking using biochemical and optical microscopy methods. The dynamic relationship between the insulin-regulated and general endosomal pathways will be defined using an HRP-mediate fluorescence quenching protocol. The concepts developed in aim 1 will be used in the other aims to further define the molecular mechanism of insulin-regulated trafficking. In aim 2 we will use dominant-inhibitors to determine whether proteins that regulate traffic from endosomes to the cell surface also function along the insulin-regulated pathway. In this aim we will also investigate the machinery that sorts GLUT4 from endosomes by determining whether the LL/acidic cluster motif that targets proteins to an insulin-regulated pathway in fibroblasts also functions in adipocytes. In aim 3 we will use the two-step insulin-regulated retention model as a conceptual framework for studies of how effectors of insulin-signal transduction recruit GLUT4 to the cell surface. The information derived from the studies of aims 1 and 2 is critical for understanding how the downstream effectors of the insulin receptor function to induce translocation of GLUT4. In aim 4 we will identify the proteins of the insulin-regulated pathway in adipocytes using the HRP-mediated compartment density shift method. A long-term objective is to determine the roles that these proteins play in insulin-regulated trafficking in adipocytes.